Supplementary Figure 3: PEG-IgG3 isolated from serum of HIV-viremic individuals with high-intensity IgG3 on IgM⁺ B cells binds to B cells of HIV-negative individuals.

(a) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with PEG-IgG3 isolated from sera of HIV-viremic individuals with low- or high-intensity IgG3 on IgM⁺ B cells. Bottom: cumulative data from paired sera of HIV-viremic individuals with high (yellow; n = 12) or low (light green; n = 12) intensity IgG3 on IgM⁺ B cells. Heat map depicts % of IgM-expressing B cells from HIV-negative individuals with bound IgG3 following incubation with serum-derived PEG precipitate or bead-enriched IgG3. PEG-IgG3 isolated with serum from individuals with high-intensity IgG3 on IgM⁺ B cells bound B cells of HIV-negative individuals with a median 39%, compared to 0% from those with low-intensity IgG3 on IgM⁺ B cells (P = 0.005). For bead-enriched IgG3, the medians were 8.2% for high versus 1.8% for low-intensity IgG3 on IgM⁺ B cells (P = 0.009). P values, two-tailed Wilcoxon matched-pairs signed rank test. (b) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with PEG-IgG3 isolated from the serum of HIV-viremic individuals with low- or high-intensity IgG3 on IgM⁺ B cells. The plots show IgG3 and IgM staining for TLM and naïve B cells, defined by the expression of CD21 and CD27. (c) Comparison of calcium uptake by HIV-negative IgD⁺ (D) TLM and naïve (N) B-cell IgG3^+/– (G3) populations treated with PEG-IgG3 isolated from serum of chronically infected HIV-viremic individuals with moderate to high-intensity IgG3 on IgM⁺ B cells. Each color coded symbol represents an individual (n = 6); black horizontal bars represent medians; * P < 0.05; n.s., not significant (two-tailed Wilcoxon matched-pairs signed rank test after obtaining significance by Friedman ANOVA test on full set). Data are representative of 12 individual experiments (b).